MNAzymes and gold nanoparticles as isothermal signal amplification strategy for visual detection of miRNA.

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples.

Inorganic nanoparticles coupled to nucleic acid enzymes as analytical signal amplification tools.

Nucleic acid enzymes (NAzymes) are a class of nucleic acid molecules with catalytic activity, which can be modulated by the presence of different species such as metal ions, genetic biomarkers, small molecules or proteins, among others. NAzymes offer several important advantages for development of novel bioanalytical strategies, resulting from their functionality as specific recognition elements and as amplified analytical signal generators, making them ideal candidates for developing highly specific bioanalytical strategies for the detection of a wide variety of targets. When coupled with the exceptional features of inorganic nanoparticles (NPs), the sensitivity of the assays can be significantly improved, allowing the detection of targets using many different detection techniques including visual readout, spectrophotometry, fluorimetry, electrochemiluminescence, voltammetry, and single-particle inductively coupled plasma-mass spectrometry. Here we provide an overview of the fundamentals of novel strategies developed to achieve analytical signal amplification based on the use of NAzymes coupled with inorganic NPs. Some representative examples of such strategies for the highly sensitive detection of different targets will be presented, including metal ions, proteins, DNA- or RNA-based biomarkers, and small molecules or microorganisms. Furthermore, future prospective challenges will be discussed.

Visual detection of microRNA146a by using RNA-functionalized gold nanoparticles.

Gold nanoparticles of different sizes have been synthesized and surface-functionalized with selected RNA probes in order to develop a rapid, low-cost and sensitive method for detection of microRNA146a (miR146a). The strategy is based on the change of colour that can be observed visually after aggregation of the RNA modified-gold nanoparticles (AuNPs) in presence of miR146a. Experimental conditions have been carefully selected in order to obtain a good sensitivity that allows to perform visual detection of microRNA at the nM level, achieving a detection limit of 5 nM. Good repeatability and selectivity versus other sequences that only differ from miR146a in 3 bases was achieved. miR146a has been described as one of the main microRNA involved in the immune response of bovine mastitis, being expressed in tissue, blood and milk samples. The method was successfully applied to the detection of miR146a in raw cow milk samples. The present scheme constitutes a rapid and low-cost alternative to perform highly sensitive detection of microRNA without the need of instrumentation and amplification steps for the early detection of bovine mastitis in the agrofood industry. Graphical abstract Schematic representation of the assay based on aggregation of RNA-modified gold nanoparticles (blue) in presence of microRNA146a generating a dark blue spot onto a solid support, versus a pink spot observed in absence of miR146a due to dispersed gold nanoparticles (red).